Compositions and methods of use of orf1358 from beta-hemolytic streptococcal strains

ABSTRACT

The present invention relates to polynucleotides encoding  Streptococcus  group C and G polypeptides and their use in immunogenic compositions. The invention also relates to immunogenic compositions comprising polypeptides encoded by those polynucleotides. In addition, the invention relates to methods of inducing an immune response in mammals against beta hemolytic  Streptococcus  or beta hemolytic  Streptococcus  infection using immunogenic compositions of the  Streptococcus  group C and G polypeptides and polynucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Provisional Application No.61/074,251, filed Jun. 20, 2008. The contents of this application arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to polynucleotides obtained from Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis and the polypeptidesencoded by such polynucleotides.

BACKGROUND OF THE INVENTION

The beta-hemolytic streptococcus species are important pathogensresponsible for numerous human diseases ranging from superficialinfections to more severe illnesses. They include species fromserological groups A, B, C and G. Group A Streptococcus bacteria(Streptococcus pyogenes) are accountable for most cases of illness andcan result in non-invasive disease such as pharyngitis, scarlet fever,impetigo, cellulitis or erysipelas. Some Streptococcus strains can leadto more severe invasive infections such as toxic shock syndrome,necrotizing fasciitis and septicemia. Additionally, complications ofsurface infections can result in immune-mediated sequelae. Lancefield'sGroup B streptococcus (Streptococcus agalactiae) is the predominantcause of neonatal sepsis in neonates and can cause pneumonia in elderlypatients. Streptococcal groups C and G were initially recognized asanimal pathogens but in recent years have been shown to have a strongpotential for human disease. Illness caused by Streptococcal groups Cand G generally presents itself similarly as in Group A streptococcusbut has not been shown to lead to immune-mediated sequelae. Group C andG streptococci are often present in patients with underlying healthproblems, are of importance for elderly patients and are dispersed amongseveral streptococcal species.

SUMMARY OF THE INVENTION

The invention is based on the discovery of novel Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis polynucleotidescorresponding to the Streptococcus pyogenes open reading frame 1358(ORF1358). The invention encompasses the polypeptides encoded by suchpolynucleotides.

In one embodiment, the invention provides an isolated polypeptide thatcomprises at least a fragment of the amino acid sequence set forth inSEQ ID NO:31, which is a consensus sequence of the various novel ORF1358 sequences obtained from Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharynges. In some embodiments, the isolatedpolypeptide comprises an amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, or a fragmentthereof. In some embodiments, the isolated polypeptide comprises anamino acid sequence that is at least 97.5%, 98, or 99% identical to theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, theisolated polypeptide has zinc-binding activity. In some embodiments theisolated polypeptide comprises an amino acid sequence that is at least90%, 95%, 97.5%, 98, or 99% identical to the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, Or SEQ ID NO:32.

In one embodiment, the invention provides an isolated polynucleotidethat encodes a polypeptide comprising an amino acid sequence set forthin SEQ ID NO:31, or a fragment thereof. In some embodiments, theisolated polynucleotide encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32, or a fragment thereof. In someembodiments, the isolated polynucleotide comprises a nucleotide sequenceset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, or a fragment thereof. In some embodiments, the isolatedpolynucleotide encodes a polypeptide comprising an amino acid sequencethat is at least 90%, 95%, 97.5%, 98, or 99% identical to the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, the isolatedpolynucleotide comprises a nucleotide sequence that is at least 90%,95%, or 99% identical to the polynucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31. In someembodiments, the isolated polynucleotide encodes a zinc bindingpolypeptide. In some embodiments, the polynucleotide is operably linkedto a regulatory element. In some embodiments, the regulatory elementcomprises an inducible promoter and/or a constitutive promoter.

In one embodiment, the invention provides an antibody that specificallybinds to at least a fragment of at least one Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis ORF1358 isolatedpolypeptide. In some embodiments, the antibody binds an isolatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32. Theantibody may be a monoclonal antibody or a polyclonal antibody.

In one embodiment, the invention provides a kit comprising an ORF 1358isolated polypeptide or a fragment thereof whose amino acid sequence iselucidated from Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharynges. In some embodiments, the kit comprises an isolatedpolypeptide comprising an amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32, ora fragment thereof. In some embodiments, the kit comprises apolynucleotide vector expressing a polypeptide, or a fragment thereof,encoded by ORF 1358 of Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharynges. In some embodiments, the kit comprises apolynucleotide vector expressing a polypeptide which comprises the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32, or a fragment thereof.

In one embodiment, the invention provides a polynucleotide vectorexpressing a Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis ORF1358 polypeptide. In some embodiments, the isolatedpolynucleotide vector expresses a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, thepolynucleotide vector comprises an isolated polynucleotide that encodesa polypeptide comprising the amino acid sequence set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32.In some embodiments, the polynucleotide vector comprises the nucleotidesequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or SEQ ID NO:31. In some embodiments, the polynucleotide vectorcomprises a polynucleotide that encodes a polypeptide comprising anamino acid sequence that is at least 90%, 95%, 97.5%, 98%, or 99%identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32. Insome embodiments, the polynucleotide vector comprises an isolatedpolynucleotide that encodes a polypeptide with an amino acid sequencethat is at least 90%, 95%, 97.5%, 98%, or 99% identical to the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, thepolynucleotide vector comprises an isolated polynucleotide that encodesa zinc binding polypeptide. In some embodiments, the polynucleotidevector comprises an isolated polynucleotide comprising a regulatorysequence operably linked to the isolated polynucleotide. In someembodiments, the polynucleotide vector comprises a regulatory element,which may be a constitutive promoter or an inducible promoter. In someembodiments, the polynucleotide vector is a plasmid, a viral vector, oran expression vector.

In one embodiment, the invention provides an immunogenic compositioncomprising an isolated Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis ORF 1358 polypeptide. In some embodiments, theimmunogenic composition comprises a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32.

In one embodiment, the invention provides an immunogenic compositioncomprising a Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis isolated polynucleotide encoding an ORF1358polypeptide. In some embodiments, the immunogenic composition comprisesa polynucleotide comprising the nucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31.

In one embodiment, the invention provides a method for inducing animmune response to beta hemolytic Streptococcus or beta hemolyticStreptococcus infection in a mammal comprising administering to themammal an immunogenic composition comprising a Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis isolated ORF1358polypeptide.

In one embodiment, the invention provides an ex-vivo host cellexpressing an isolated polypeptide encoded by Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis ORF1358. In someembodiments, the host cell expresses a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, the host cellcomprises a polynucleotide vector comprising an isolated polynucleotidethat encodes a polypeptide comprising the amino acid sequence set forthin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQID NO:32. In some embodiments, the host cell comprises a polynucleotidevector comprising the nucleotide sequence set forth in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31. In some embodiments,the host cell comprises a polynucleotide vector comprising apolynucleotide that encodes a polypeptide comprising an amino acidsequence that is at least 90%, 95%, 97.5%, 98%, or 99% identical to theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, the hostcell comprises a polynucleotide vector comprising an isolatedpolynucleotide encoding a polypeptide with an amino acid sequence thatis at least 90%, 95%, 97.5%, 98%, or 99% identical to the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, the host cellcomprises a polynucleotide vector comprising an isolated polynucleotideencoding a zinc binding polypeptide. In some embodiments, the host cellcomprises a polynucleotide vector comprising an isolated polynucleotidecomprising a regulatory sequence operably linked to the isolatedpolynucleotide. In some embodiments, the host cell comprises apolynucleotide vector comprising a regulatory element, which may be aconstitutive promoter or an inducible promoter. In some embodiments, thehost cell comprises a polynucleotide vector that is a plasmid, a viralvector, or an expression vector. In some embodiments the host cell isselected from a bacterium, a mammalian cell, an insect cell, or a yeastcell.

In one embodiment, the invention provides a kit comprising apolynucleotide or polypeptide comprising a Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis ORF1358 polynucleotide orpolypeptide.

In one embodiment, the invention provides a method for treating a betahemolytic Streptococcus infection in a mammal comprising administering atherapeutically effective amount of an antibody that specifically bindsto at least one isolated polypeptide comprising a polypeptide encoded byStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358. In some embodiments, the method uses an antibody that binds anisolated polypeptide comprising the amino acid sequence set forth in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32. Theantibody used in the method may be a monoclonal antibody or a polyclonalantibody. In some embodiments, the beta hemolytic Streptococcusinfection is treated in a human.

In one embodiment, the invention provides the use of an isolatedpolypeptide comprising a polypeptide encoded by Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF 1358 in themanufacture of a medicament useful in the prophylactic treatment of abeta hemolytic Streptococcus infection in a mammal. In some embodiments,the medicament is useful in a prophylactic treatment in a human.

In one embodiment, the invention provides a medicament useful in theprophylactic treatment of a beta hemolytic Streptococcus infection in amammal. In some embodiments, the medicament uses an isolatedStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngispolynucleotide comprising an ORF1358 polypeptide. In some embodiments,the medicament uses an isolated polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. In some embodiments, themedicament uses an isolated Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis polynucleotide comprising an ORF1358polynucleotide. In some embodiments, the medicament uses an isolatedpolynucleotide comprising the nucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31. In someembodiments, the medicament uses a polynucleotide vector comprising aStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polynucleotide. In some embodiments, the medicament uses anantibody that specifically binds to at least one isolated polypeptidecomprising a polypeptide encoded by Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis ORF1358. In some embodiments, themedicament uses an antibody that specifically binds an isolatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. The medicamentmay use a monoclonal or a polyclonal antibody. In some embodiments, themammal that the medicament is used in is a human.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the nucleotide sequence of orf 1358 in a Streptococcusdysgalactiae subsp. equisimilis.

SEQ ID NO:2 is the amino acid sequence encoded by orf 1358 in aStreptococcus dysgalactiae subsp. equisimilis of SEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence of orf 1358 in a Streptococcusintermedius.

SEQ ID NO:4 is the amino acid sequence encoded by orf 1358 in aStreptococcus intermedius of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence of orf 1358 in a Streptococcusconstellatus subsp. constellatus.

SEQ ID NO:6 is the amino acid sequence encoded by orf 1358 in aStreptococcus constellatus subsp. constellatus of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence of orf 1358 in a Streptococcusanginosus.

SEQ ID NO:8 is the amino acid sequence encoded by orf 1358 inStreptococcus anginosus of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence of orf 1358 in a Streptococcusdysgalactiae subsp. equisimilis.

SEQ ID NO:10 is the amino acid sequence encoded by orf 1358 in aStreptococcus dysgalactiae subsp. equisimilis of SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide sequence of orf 1358 in Streptococcusconstellatus subsp pharyngis.

SEQI D NO:12 is the amino acid sequence encoded by orf 1358 inStreptococcus constellatus subsp. pharyngis of SEQ ID NO:11.

SEQ ID NO:13 is the consensus amino acid sequence obtained by aligningthe polypeptide sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, and12.

SEQ ID NO:14 is the nucleotide sequence of primer D1358 F1.

SEQ ID NO:15 is the nucleotide sequence of primer D1358 F3.

SEQ ID NO:16 is the nucleotide sequence of primer D1358 F5.

SEQ ID NO:17 is the nucleotide sequence of primer D1358 R2.

SEQ ID NO:18 is the nucleotide sequence of primer D1358 R3.

SEQ ID NO:19 is the nucleotide sequence of primer D1358 R5.

SEQ ID NO:20 is the nucleotide sequence of primer 1358 F.

SEQ ID NO:21 is the nucleotide sequence of primer 1358 R.

SEQ ID NO:22 is the amino acid sequence of the Streptococcus pyogeneshigh-affinity zinc uptake system protein znuA precursor having NCBI gi50902983.

SEQ ID NO:23 is the amino acid sequence of the Streptococcus agalactiae2603V/R zinc binding adhesion lipoprotein having NCBI gi 22536713.

SEQ ID NO:24 is the amino acid sequence of the polypeptide encoded by aStreptococcus agalactiae ORF1358.

SEQ ID NO:25 is the nucleotide sequence of orf 1358 in a Streptococcusdysgalactiae subsp. equisimilis.

SEQ ID NO:26 is the amino acid sequence encoded by orf 1358 in aStreptococcus dysgalactiae subsp. equisimilis of SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence of orf 1358 in a Streptococcusdysgalactiae subsp. equisimilis.

SEQ ID NO:28 is the amino acid sequence encoded by orf 1358 in ain aStreptococcus dysgalactiae subsp. equisimilis of SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence of orf 1358 in a Streptococcusanginosus.

SEQ ID NO:30 is the amino acid sequence encoded by orf 1358 in aStreptococcus anginosus of SEQ ID NO:29.

SEQ ID NO:31 is the nucleotide sequence of orf 1358 in a Streptococcusconstellatus subsp. constellatus.

SEQ ID NO:32 is the amino acid sequence encoded by orf 1358 in aStreptococcus constellatus subsp. constellatus of SEQ ID NO:31.

DETAILED DESCRIPTION

The invention describes novel polynucleotides obtained fromStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, and Streptococcus constellatus subsp. pharyngis(Streptococcal C+G) strains corresponding to Streptococcus pyogenes openreading frame 1358 (ORF1358). Polynucleotide and amino acid sequencesfor ORF 1358 are provided in published International patent applicationnumber WO 02/083859. The novel ORF1358 polynucleotides encode novelpolypeptides. These polynucleotides and polypeptides may be used inimmunogenic compositions to induce an immune response to beta hemolyticstreptococcus or beta hemolytic streptococcus infection in a mammal.

The terms “polynucleotide”, and “nucleic acid”/“nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesconnected by phosphodiester linkages. A “polynucleotide” may be aribonucleic acid (RNA) or deoxyribonucleic acid (DNA) polymer that issingle- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may comprise one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof. Nucleotide bases are indicatedhereinafter by a single letter code: adenine (A), guanine (G), thymine(T), cytosine (C), inosine (I) and uracil (U).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide.

The term “Isolated” means altered “by the hand of man” from the naturalstate. If a composition or substance occurs in nature, in order for itto be considered “Isolated” it must have been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living animal is not “isolated,” butthe same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated,” as the term is employedhereinafter. Isolated polynucleotides or isolated polypeptides may bepurified from a cell in which they naturally occur. Conventional nucleicacid and polypeptide purification methods known to skilled artisans maybe used to obtain isolated polynucleotides or polypeptides disclosedherein.

The term “operably linked” refers to the association of nucleic acidsequences on a single polynucleotide so that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence when it is capable of affecting the expression of thatcoding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The ORF1358 polynucleotides and ORF1358 polypeptides described hereinmay be obtained using standard cloning and screening techniques. TheStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, and Streptococcus constellatus subsp. pharyngisORF1358 polynucleotides may be obtained, for example, from genomic DNA,from a cDNA library derived from mRNA, from a genomic DNA library, orcan be synthesized using well known and commercially availabletechniques, such as e.g. by PCR from a cDNA library or via RT-PCR(reverse transcription-polymerase chain reaction).

The term “recombinant” means, for example, that a polynucleotide is madeby an artificial combination of two otherwise separated polynucleotidesegments, e.g., by chemical synthesis or by the manipulation of isolatedpolynucleotides using genetic engineering techniques. A “recombinant DNAconstruct” comprises any of the isolated polynucleotides of the presentinvention operably linked to at least one regulatory element.

In one embodiment, the invention provides an isolated polypeptide thatcomprises the amino acid sequence set forth in SEQ ID NO:13. The aminoacid sequence set forth in SEQ ID NO:13 is the consensus sequenceobtained after aligning the amino acid sequences encoded by theStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, and Streptococcus constellatus subsp. pharyngispolynucleotide sequences ORF1358 and set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32.

In one embodiment, the invention provides isolated polynucleotidesencoding polypeptides comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, Or SEQ ID NO:32, or fragments thereof.Encompassed herein are polynucleotides that differ from thepolynucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQID NO:29, or SEQ ID NO:31 due to degeneracy of the genetic code. Thesepolynucleotides encode polypeptides comprising the same function as thepolypeptide encoded by Streptococcus pyogenes ORF1358. The polypeptidesmay comprise zinc binding activity.

Orthologues and allelic variants of the Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, andStreptococcus constellatus subsp. pharyngis ORF1358 polynucleotides canreadily be identified using methods well known in the art. Allelicvariants and orthologues of the ORF1358 polynucleotides can comprise anucleotide sequence that is typically at least about 90-95% or moreidentical to any one or more of the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or afragments thereof. The allelic variants and orthologues of ORF1358polynucleotides can encode a polypeptide that comprises an amino acidsequence that is at least90%, 95%, or 97.5% identical to the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32. Such polynucleotides can readilybe identified as being able to hybridize under stringent conditions, toat least a fragment from any one or more of the polynucleotides havingthe nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, or a fragment thereof.

Moreover, the allelic variants and orthologues of ORF1358polynucleotides can comprise only a fragment of the coding region of aStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, and Streptococcus constellatus subsp. pharyngisORF1358 polynucleotide or gene, such as a fragment of a polynucleotideset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, or SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQID NO:31. In certain embodiments, such fragments encode immunogenicfragments.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying related polynucleotides andpolypeptides. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments were also performedusing BLAST (Altschul S F, Madden T L, Schaffer A A, et al. Gapped BLASTand PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Research. Sep. 1, 1997;25(17):3389-3402).

The ORF1358 polynucleotides of the invention may be used, for example,for the production of recombinant polypeptides for inclusion inimmunogenic compositions. For the production of recombinantpolypeptides, the polynucleotide may include the coding sequence for themature polypeptide, by itself, or the coding sequence for the maturepolypeptide linked with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro-proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can belinked to the coding sequence (see Gentz et al., Proc. Natl. Acad. Sci.USA, 86:821-824, 1989). The polynucleotide may also contain sequences 5′and/or 3′ of the coding sequence, such as transcribed sequences,non-translated sequences, splicing signals, and polyadenylation signals.

In certain embodiments, the polynucleotide sequence information providedherein allows for the preparation of relatively short DNA (or RNA)oligonucleotide sequences having the ability to specifically hybridizeto nucleotide sequences of the selected polynucleotides disclosedherein. The term “oligonucleotide” as used herein is defined as amolecule comprising two or more deoxyribonucleotides or ribonucleotides,usually more than three (3), and typically more than ten (10) and up toone hundred (100) or more (although preferably between twenty andthirty). The exact size will depend on many factors, which in turndepends on the ultimate function or use of the oligonucleotide. Thus, insome embodiments, nucleic acid probes of an appropriate length areprepared based on a selected nucleotide sequence, e.g., a sequence suchas that shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQID NO:31. The ability of such nucleic acid probes to specificallyhybridize to a polynucleotide encoding a Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis ORF1358 polypeptide lendsthem particular utility in a variety of embodiments. In someembodiments, the probes can be used in a variety of assays for detectingthe presence of complementary sequences in a given sample. These primersmay be generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, or a combination thereof. Thesequence of such primers is designed using a polynucleotide describedherein for use in detecting, amplifying or mutating a defined segment ofa polynucleotide that encodes a Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis ORF1358 polypeptide from prokaryotic cellsusing polymerase chain reaction (PCR) technology.

In some embodiments, the polynucleotides described herein may be used incombination with an appropriate label for detecting hybrid formation. Awide variety of appropriate labels are known in the art, includingradioactive, enzymatic, or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in one of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:14 through SEQ IDNO:21, or a fragment thereof, may be used as hybridization probes forcDNA and genomic DNA, or as primers for a nucleic acid amplification(PCR) reaction, to isolate full-length cDNAs and genomic clones encodingpolypeptides described herein and to isolate cDNA and genomic clones ofother genes (including genes encoding homologs and orthologs fromspecies other than Streptococcus dysgalactiae) that have a high sequencesimilarity to the polynucleotide sequences set forth in of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or a fragmentthereof. Typically these nucleotide sequences are from at least about90% identical to at least about 99% identical to that of the referencepolynucleotide sequence. The probes or primers will generally compriseat least 15 nucleotides, at least 30 nucleotides or at least 50nucleotides.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs. For examplethose methods based on the method of Rapid Amplification of cDNA ends(RACE) (See Frohman et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002,1988). Modifications of this technique, exemplified by the Marathon™technology (Clontech, Mountain View, Calif.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs are prepared from mRNA extracted from a chosen tissueand an “adaptor” sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the cDNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length cDNAconstructed either by joining the product directly to the existing cDNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

In one embodiment, the present invention provides isolated and purifiedStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptides for use in immunogenic compositions. An ORF1358polypeptide used in an immunogenic composition of the invention may be arecombinant polypeptide.

A Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptide used in an immunogenic composition of the presentinvention encompasses a polypeptide that comprises an amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, Or SEQ ID NO:32, or a fragment thereof; functionaland non-functional naturally occurring variants or biologicalequivalents of said polypeptides; recombinantly produced variants orbiological equivalents of said polypeptides; orthologues, or allelicvariants of said polypeptides.

Biological equivalents or variants of Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis ORF1358 include both functional andnon-functional Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis ORF1358 polypeptides. Functional biological equivalentsor variants include naturally occurring amino acid sequence variants ofa Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptide that maintains the ability to elicit animmunological or antigenic response in a subject. Functional variantstypically contain conservative substitutions of one or more amino acidsof one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, Or SEQ ID NO:32; or substitutions, deletions or insertionsof non-critical residues in non-critical regions of the polypeptide.

In some embodiments, modifications and changes can be made in thestructure of a polypeptide of the present invention and still obtain amolecule having the same antigenicity as the unchanged Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide. Forexample, certain amino acids can be substituted for other amino acids ina sequence without appreciable loss of antigenicity. Because it is theinteractive capacity and nature of a polypeptide that defines thatpolypeptide's biological functional activity, certain amino acidsequence substitutions can be made in a polypeptide sequence (or itsunderlying DNA coding sequence) and nevertheless obtain a polypeptidewith like properties.

In making changes to obtain orthologues or allelic variants, thehydropathic index of amino acids can be considered. The importance ofthe hydropathic amino acid index in conferring interactive biologicfunction on a polypeptide is generally understood in the art (Kyte andDoolittle, J Mol Biol, 157: p. 105-132, 1982). It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still result in a polypeptide withsimilar biological activity. Each amino acid has been assigned ahydropathic index on the basis of its hydrophobicity and chargecharacteristics. Those indices are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9) tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is generally accepted in the art that the relative hydropathiccharacter of the amino acid residue determines the secondary andtertiary structure of the resultant polypeptide, which in turn definesthe interaction of the polypeptide with other molecules, such asenzymes, substrates, receptors, antibodies, antigens, and the like. Itis known in the art that an amino acid can be substituted by anotheramino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In some embodiments,polynucleotides encoding ORF1358 polypeptide may comprise substitutedamino acids whose hydropathic indices are within ±2. In someembodiments, the hydrophobic indices are within ±1, and someembodiments, the hydrophobic indices are within ±0.5.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporatedhereinafter by reference, states that the greatest local averagehydrophilicity of a polypeptide, as governed by the hydrophilicity ofits adjacent amino acids, correlates with its immunogenicity andantigenicity.

A “variant,” as the term is used herein is a polynucleotide or apolypeptide that differs from a reference polynucleotide or referencepolypeptide respectively, while retaining at least one essentialproperty. A typical variant of a polynucleotide differs in nucleotidesequence from a reference polynucleotide. Changes in the nucleotidesequence of the variant may or may not alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencepolynucleotide, as discussed below. A typical variant of a polypeptidediffers in amino acid sequence from a reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant polypeptide are closely similar overall and,in many regions, identical. A variant polypeptide and its referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, or deletions, in any combination. Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. A variant of a polynucleotide or polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques or by direct synthesis.

For recombinant production of polypeptides, host cells are geneticallyengineered to incorporate expression systems, portions thereof, orpolynucleotides of the invention. Polynucleotides comprising ORF1358 canbe introduced into host cells e.g. by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). These methods include e.g. calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, ultrasound, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction,and infection.

Representative examples of suitable host cells include bacterial cells(e.g., streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells), yeast cells (e.g., Pichia, Saccharomyces), mammaliancells (e.g., vero, Chinese hamster ovary, chick embryo fibroblasts, BHKcells, human SW13 cells), and insect cells (e.g., Sf9, Sf21).

The recombinantly-produced polypeptides may be recovered and purifiedfrom recombinant cell cultures by well-known methods, including highperformance liquid chromatography, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and lectinchromatography.

Any one or more systems may be used to express and produce theStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptides in a heterologous cell system. Such systemsinclude, among others, chromosomal, episomal and virus-derived systems.Vectors may be derived from bacterial plasmids, attenuated bacteria,bacteriophage, transposons, yeast episomes, insertion elements, yeastchromosomal elements, or viruses. Vectors may be obtained from virusessuch as vaccinia and other poxviruses, sindbis, adenovirus,baculoviruses, papova viruses (such as SV40), fowl pox viruses,pseudorabies viruses, retroviruses, alphaviruses (such as Venezuelanequine encephalitis virus (U.S. Pat. No. 5,643,576)), nonsegmentednegative-stranded RNA viruses such as vesicular stomatitis virus (U.S.Pat. No. 6,168,943). Vectors may also be derived from combinationsthereof, such as those derived from plasmid and bacteriophage geneticelements, such as cosmids and phagemids. The expression systems shouldinclude control regions that regulate as well as engender expression,such as promoters and other regulatory elements (such as apolyadenylation signal). Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

In one embodiment, the present invention provides expression vectorsexpressing Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptides for use in immunogenic compositions. The expressionvectors comprise ORF1358 polynucleotides encoding polypeptidescomprising an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, Or SEQ ID NO:32, or a fragmentthereof. Alternatively, the expression vectors comprise a polynucleotidecomprising a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or a fragment thereof. In otherembodiments, the expression vectors of the invention comprise apolynucleotide operatively linked to an enhancer-promoter. In stillother embodiments, the expression vectors comprise a polynucleotideoperatively linked to a prokaryotic promoter. Alternatively, theexpression vectors comprise a polynucleotide operatively linked to anenhancer-promoter that is a eukaryotic promoter. The expression vectorsfurther may comprise a polyadenylation signal that is positioned 3′ ofthe carboxy-terminal amino acid and within a transcriptional unit of theencoded polypeptide.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters that cause a nucleic acid fragment to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity Commonly used promoters are derived fromviruses such as polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells see chapters 16 and 17 of Sambrook et al., “MolecularCloning: A Laboratory Manual” 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989,incorporated hereinafter by reference. In certain instances, theexpression vector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., Genes Dev, 1: p. 268-277,1987), lymphoid-specific promoters (Calame and Eaton, Adv Immunol, 43:p. 235-275, 1988), in particular, promoters of T cell receptors (Winotoand Baltimore, EMBO J, 8: p. 729-733, 1989) and immunoglobulins (Banerjiet al., Cell, 33: p. 729-740, 1983), (Queen and Baltimore, Cell, 33: p.741-748, 1983), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle, PNAS, 86: p. 5473-5477, 1989),pancreas-specific promoters (Edlund et al., Science, 230: p. 912-916,1985), and mammary gland-specific promoters (e.g., milk whey promoter;U.S. Pat. No. 4,873,316 and International Application EP 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss, Science, 249: p. 374-379,1990) and the a-fetoprotein promoter (Campes and Tilghman, Genes Dev, 3:p. 537-546, 1989).

Also provided herein are recombinant expression vectors comprisingpolynucleotides encoding at least a portion of a Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptidescloned into the expression vector in an antisense orientation. That is,the DNA molecule is operatively linked to a regulatory sequence in amanner which allows for expression (by transcription of the DNAmolecule) of an RNA molecule which is antisense to the Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide mRNA.Regulatory sequences operatively linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types. Forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced.

The recombinant expression vectors described herein may be inserted intoany suitable host cell. The terms “host cell” and “recombinant hostcell” are used interchangeably herein. It is understood that such termsrefer not only to the particular subject cell, but also to the progenyor potential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, a Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptide can be expressed in bacterial cells (such as E.coli), insect cells (such as Sf9, Sf21), yeast cells, or mammalian cells(such as Chinese hamster ovary cells (CHO), VERO, chick embryofibroblasts, BHK cells or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA is introduced into prokaryotic or eukaryotic cells viaconventional transformation, infection or transfection techniques. Asused herein, the terms “transformation” and “transfection” are intendedto refer to a variety of art-recognized techniques for introducingforeign nucleic acid (e.g., DNA) into a host cell, including calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, ultrasound or electroporation. Suitablemethods for transforming or transfecting host cells can be found, forexample, in Sambrook, et al. (“Molecular Cloning: A Laboratory Manual”2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

A host cell described herein, such as a prokaryotic or a eukaryotic hostcell in culture, is used to produce (i.e., express) a Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide.Accordingly, also described herein are methods for producing apolypeptide using such host cells. In one embodiment, the methodcomprises culturing the host cell (into which a recombinant expressionvector encoding an ORF1358 polypeptide has been introduced) in asuitable medium until the polypeptide is produced. In anotherembodiment, the method further comprises isolating the ORF1358polypeptide from the medium or the host cell.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins.Constitutive promoters include, for example, lambda PL, spc ribosomaland beta-lactamase. Inducible promoters include, for example, arabinose,lac, tac and maltose binding protein. Fusion vectors add a number ofamino acids to a protein encoded therein, usually to the amino terminusof the recombinant protein. Such fusion vectors typically serve threepurposes: to increase expression of recombinant protein; to increase thesolubility of the recombinant protein; and to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. The invention also provides vectors (e.g., expressionvectors, sequencing vectors, cloning vectors) which comprise at leastone polynucleotide of the invention, host cells which are geneticallyengineered with vectors of the invention, and production of polypeptidesof the invention by recombinant techniques. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the invention.

Expression vectors useful to express Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis ORF1358 polypeptides are viral vectors,such as lentiviruses, retroviruses, herpes viruses, adenoviruses,adeno-associated viruses, vaccinia virus, baculovirus, and otherrecombinant viruses with desirable cellular tropism. Thus, a geneencoding a functional or mutant protein or polypeptide, or fragmentthereof can be introduced in vivo, ex vivo, or in vitro using a viralvector or through direct introduction of DNA. Expression in targetedtissues can be effected by targeting the transgenic vector to specificcells, such as with a viral vector or a receptor ligand, or by using atissue-specific promoter, or both. Targeted gene delivery is describedin PCT Publication Number WO 95/28494.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (e.g., Millerand Rosman, BioTechniques, 1992, 7:980-990). Preferably, the viralvectors are replication-defective, that is, they are unable to replicateautonomously in the target cell. Preferably, the replication defectivevirus is a minimal virus, i.e., it retains only the sequences of itsgenome, which are necessary for encapsulating the genome to produceviral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as,for example, herpes simplex virus (HSV), papillomavirus, Epstein Barrvirus (EBV), adenovirus, adeno-associated virus (AAV), and the like.Defective viruses, which entirely or almost entirely lack viral genes,are preferred. A defective virus is not infective after introductioninto a cell. Use of defective viral vectors allows for administration tocells in a specific, localized area, without concern that the vector caninfect other cells. Thus, a specific tissue can be specificallytargeted. Examples of particular vectors include, but are not limitedto, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec.Cell. Neurosci., 1991, 2:320-330), defective herpes virus vector lackinga glycoprotein L gene, or other defective herpes virus vectors (PCTPublication Numbers WO 94/21807 and WO 92/05263); an attenuatedadenovirus vector, such as the vector described by Stratford-Perricaudetet al. (J. Clin. Invest., 1992, 90:626-630; see also La Salle et al.,Science, 1993, 259:988-990); and a defective adeno-associated virusvector (Samulski et al., J. Virol., 1987, 61:3096-3101; Samulski et al.,J. Virol., 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol., 1988,8:3988-3996).

The polypeptides of the invention, including those comprising the aminoacid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:13, their fragments,and analogs thereof, or cells expressing them, can also be used asimmunogens to produce antibodies immunospecific for the polypeptides ofthe invention. The invention includes antibodies immunospecific forStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptides, the use of such antibodies to detect the presenceof, or measure the quantity or concentration of Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptides in acell, a cell or tissue extract, or a biological fluid, or for treatmentof Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisinfection.

The antibodies of the invention include polyclonal antibodies,monoclonal antibodies, chimeric antibodies, and anti-idiotypicantibodies. Polyclonal antibodies are heterogeneous populations ofantibody molecules derived from the sera of animals immunized with anantigen. Monoclonal antibodies are a substantially homogeneouspopulation of antibodies to specific antigens. In general, antibodiescan be made, for example, using traditional hybridoma techniques (Kohlerand Milstein Nature, 256: 495-499, 1975), recombinant DNA methods (U.S.Pat. No. 4,816,567), or phage display using antibody libraries (Clacksonet al. Nature 352: 624-628, 1991; Marks et al. J. Mol. Biol. 222:581-597, 1991). For additional antibody production techniques, seeAntibodies: A Laboratory Manual, eds. Harlow and Lane, Cold SpringHarbor Laboratory, 1988. The present invention is not limited to anyparticular source, method of production, or other specialcharacteristics of an antibody.

Intact antibodies are immunoglobulins (Ig), and they typically aretetrameric glycpsylated proteins composed of two light chains (˜25 kDaeach) and two heavy chains (˜50 kDa each). Light chains are classifiedinto two isotypes (λ and κ), and heavy chains are classified into fiveisotypes (A, D, E, G, and M). Some heavy chain isotypes are furtherdivided into isotype subclasses, e.g., IgG₁, IgG₂, IgG₃, and IgG₄.

The domain and three dimensional structures of different antibodies areknown in the art (Harlow and Lane, supra). In brief, the light chain iscomposed of a constant domain (C_(L)) and an N-terminal variable domain(V_(L)). The heavy chain is composed of three or four constant domains(C_(H)), a hinge region, and a N-terminal variable domain (V_(H)). TheC_(H) adjacent to the V_(H) domain is designated C_(H1). The V_(H) andV_(L) domains contain four regions of conserved sequence calledframework (FR) regions (FR1, FR2, FR3, and FR4), which form a scaffoldfor three regions of hypervariable sequence called complementaritydetermining regions (CDR). The CDRs (CDR1, CDR2, and CDR3) contain mostof the antibody amino acids that specifically recognize and bindantigen. Heavy chain CDRs are denoted H1, H2, and H3, while light chainCDRs are denoted L1, L2, and L3.

The Fab fragment (Fragment antigen-binding) consists of V_(H)-C_(H1) andV_(L)-C_(L) domains covalently linked by a disulfide bond between theconstant regions. The F_(v) fragment is smaller and consists of V_(H)and V_(L) domains non-covalently linked. To overcome the tendency ofnon-covalently domains to dissociate, a single chain F_(v) fragment(scF_(v)) can be constructed. The scF_(v) contains a flexiblepolypeptide that links the (1) C-terminus of V_(H) to the N-terminus ofV_(L), or the (2) C-terminus of V_(L) to the N-terminus of V_(H). A15-mer (Gly₄Ser)₃ peptide may be used as a linker, but other linkers areknown in the art.

Antibody diversity is created by use of multiple germline genes encodingvariable regions and a variety of somatic events. The somatic eventsinclude recombination of variable gene segments and diversity (D) andjoining (J) gene segments to make a complete V_(H) region and therecombination of variable and joining gene segments to make a completeV_(L) region. CDR3 (H3) is the greatest source of molecular diversitywithin an antibody sequence. H3, for example, can be as short as twoamino acid residues or greater than 26. The smallest antigen-bindingfragment is the Fv, which consists of the V_(H) and the V_(L) domains.

Anti-ORF1358 polypeptide antibodies of this invention may optionallycomprise antibody constant regions or parts thereof. For example, aV_(L) domain may be attached at its C-terminal end to a light chainconstant domain like Cκ or Cλ. Similarly, a V_(H) domain or portionthereof may be attached to all or part of a heavy chain like IgA, IgD,IgE, IgG, and IgM, and any isotype subclass. Antibody isotype such asIgG₁, IgG₂, IgG₃ or IgG₄ is determined by the CH₂ and CH₃ domains.Isotypes may be switched by changing these domains without affectingantigen binding. Constant regions are known in the art (see, forexample, Kabat et al., Sequences of Proteins of Immunological Interest,No. 91-3242, National Institutes of Health Publications, Bethesda, Md.,1991).

Chimeric antibodies are molecules, different portions of which arederived from different animal species, such as those having variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies and methods fortheir production are known in the art (Cabilly et al., Proc. Natl. Acad.Sci. USA 81 :3273-3277, 1984; Morrison et al., Proc. Natl. Acad. Sci.USA 81:6851-6855, 1984; Boulianne et al., Nature 312:643-646, 1984;Cabilly et al., European Patent Application No. 125023 (published Nov.14, 1984); Taniguchi et al., European Patent Application No. 171496(published Feb. 19, 1985); Morrison et al., European Patent ApplicationNo. 173494 (published Mar. 5, 1986); Neuberger et al., PCT ApplicationNo. WO 86/01533 (published Mar. 13, 1986); Kudo et al., European PatentApplication No. 184187 (published Jun. 11, 1986); Morrison et al.,European Patent Application No. 173494 (published Mar. 5, 1986); Sahaganet al., J. Immunol. 137:1066-1074, 1986; Robinson et al., PCT/US86/02269(published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA84:3439-3443, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218,1987; Better et al., Science 240:1041-1043, 1988).

An anti-idiotypic (anti-Id) antibody is an antibody that recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An anti-Id antibody is prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the monoclonal antibody with the monoclonal antibody to which ananti-Id is being prepared. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody byproducing an antibody to these isotypic determinants (the anti-Idantibody).

Accordingly, monoclonal antibodies generated against the polypeptides ofthe present invention may be used to induce anti-Id antibodies insuitable animals. Spleen cells from such immunized animals can be usedto produce anti-Id hybridomas secreting anti-Id monoclonal antibodies.Further, the anti-Id antibodies can be coupled to a carrier such askeyhole limpet hemocyanin (KLH) and used to immunize additional BALB/cmice. Sera from these mice will contain anti-anti-Id antibodies thathave the binding properties of the final mAb specific for a R-PTPaseepitope. The anti-Id antibodies thus have their idiotypic epitopes, or“idiotopes” structurally similar to the epitope being evaluated, such asStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngispolypeptides encoded by ORF1358.

The term “antibody” is also meant to include both intact molecules aswell as fragments such as Fab, which are capable of binding antigen. Fabfragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nucl. Med. 24:316-325, 1983). Itwill be appreciated that Fab and other fragments of the antibodiesuseful in the present invention may be used for the detection andquantitation of Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis polypeptides according to the methods for intactantibody molecules.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

The antibodies may be used in a variety of ways, e.g., for confirmationthat a protein is expressed, or to confirm where a protein is expressed.Labeled antibody (e.g., fluorescent labeling for FACS) can be incubatedwith intact bacteria and the presence of the label on the bacterialsurface confirms the location of the protein.

Other suitable methods of producing or isolating antibodies thatspecifically bind to a Group C or Group G streptococcal ORF1358polypeptide epitope can be used. In some embodiments, the recombinantantibody is selected from a peptide or protein display library such ase.g. a bacteriophage, ribosome, oligonucleotide, RNA and cDNA displaylibraries (EP368,684; PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240;PCT/GB92/00883; PCT/GB93/00605; PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; WO90/14443; WO90/14424; WO90/14430; PCT/US94/1234;WO92/18619; WO96/07754; EP614,989; WO95/16027; WO88/06630; WO90/3809;U.S. Pat. No. 4,704,692; PCT/US91/02989; WO89/06283; EP371,998;EP550,400; EP229,046; and PCT/US91/07149.) In other embodiments, therecombinant antibody is selected from a library of stochasticallygenerated peptides or proteins (U.S. Pat. Nos. 5,723,323; 5,763,192;5,814,476; 5,817,483; 5,824,514; 5,976,862; WO 86/05803; and EP590,689.) In yet other embodiments, the recombinant antibody is producedin a transgenic animal that is capable of producing a repertoire ofhuman antibodies (Nguyen et al., Microbiol. Immunol. 41:901-907, 1997;Sandhu et al., Crit. Rev. Biotechnol. 16:95-118, 1996; and Eren et al.,Immunol. 93:154-161, 1998.) Other techniques for producing recombinantantibodies include e.g. single cell antibody producing technologies suchas the selected lymphocyte antibody method (“SLAM”) (U.S. Pat. No.5,627,052), gel microdroplet and flow cytometry methods (Powell et al.,Biotechnol. 8:333-337, 1990), and B-cell selection (Steenbakkers et al.,Molec. Biol. Reports 19:125-134, 1994). These same methods can also bedeployed to improve the affinity and/or avidity of an anti-Group C orGroup G streptococcal PPI antibody to its specific binding target.

The present invention provides immunogenic compositions comprising oneor more Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngispolypeptides encoded by ORF1358. In certain embodiments, the immunogeniccompositions comprise one or more Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis polypeptides comprising an amino acidresidue sequence that is at least 97.5%, 98%, 99%, or 100% identical toSEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, and 32, and one or morephysiologically acceptable carriers.

In other embodiments, the immunogenic compositions of the inventioncomprise polynucleotides that encode the Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis ORF1358 polypeptides, andone or more physiologically acceptable carriers. In some embodiments,the immunogenic compositions comprise polynucleotides having anucleotide sequence that is at least 90%, 95%, 99%, or 100% identical toone or more of SEQ ID NOs:1, 3, 5, 7, 9,11, 25, 27, 29, or 31.

The term “immunogenic composition” as used herein refers to any type ofbiological agent in an administratable form capable of stimulating animmune response in an animal (which includes human) inoculated with theimmunogenic composition. An immune response may include induction ofantibodies and/or induction of a T-cell response. Herein, the term“protection,” when used in reference to an immunogenic composition,refers to the amelioration (either partial or complete) of any of thesymptoms associated with the disease or condition in question. Thus,protection of animals from Streptococcus or infection by a Streptococcusspecies such as Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis by the present immunogenic compositions generallyresults in a diminishing of bacterial growth and/or one or more of theclinical symptoms associated with infection by Streptococcus species,including arthritis, endocarditis, meningitis, polyserositis,bronchopneumonia, meningitis, permanent hearing loss, and septic shock.

The methods disclosed herein may include inducing an immune responseagainst one or more pathogens that include a species of Streptococcus(e.g., Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp.pharynges). For example, the methods may include inducing polyclonalantibodies against one or more pathogens that include a species ofStreptococcus that may include Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharynges. In some embodiments, the methods includeadministering to a subject (any vertebrate, including human patients andother mammals) a composition that includes an isolated Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide orpolynucleotide.

Various tests are used to assess the in vitro immunogenicity of thepolypeptides of the invention. For example, an in vitro opsonic assay isconducted by incubating together a mixture of Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus intermedius, Streptococcusconstellatus subsp. constellatus, Streptococcus anginosus, orStreptococcus constellatus subsp. pharyngis cells, heat inactivatedserum containing specific antibodies to the polypeptide in question, andan exogenous complement source. Opsonophagocytosis proceeds duringincubation of freshly isolated polymorphonuclear cells (PMN's) and theantibody/complement/Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis cell mixture. Bacterial cells that are coated withantibody and complement are killed upon opsonophagocytosis. Colonyforming units (cfu) of surviving bacteria that escape fromopsonophagocytosis are determined by plating the assay mixture. Titersare reported as the reciprocal of the highest dilution that gives ≧50%bacterial killing, as determined by comparison to assay controls.Specimens that demonstrate less than 50% killing at the lowest serumdilution tested (1:8), are reported as having an OPA (opsonophagocytosisantibody) titer of 4. The highest dilution tested is 1:2560. Sampleswith ≧50% killing at the highest dilution are repeated, beginning with ahigher initial dilution. The method described above is a modification ofGray's method (Gray, Conjugate Vaccines Supplement, p. 694-697, 1990). Atest serum control, which contains test serum plus bacterial cells andheat inactivated complement, is included for each individual serum. Thiscontrol is used to assess whether the presence of antibiotics or otherserum components are capable of killing the bacterial strain directly(i.e. in the absence of complement or PMN's). A human serum with knownopsonic titer is used as a positive human serum control. The opsonicantibody titer for each unknown serum is calculated as the reciprocal ofthe initial dilution of serum giving 50% cfu reduction compared to thecontrol without serum.

A whole cell ELISA assay is also used to assess in vitro immunogenicityand surface exposure of the polypeptide antigen, wherein the bacterialstrain of interest is coated onto a plate, such as a 96 well plate, andtest sera from an immunized animal is reacted with the bacterial cells.If any antibody, specific for the test polypeptide antigen, is reactivewith a surface exposed epitope of the polypeptide antigen, it can bedetected by standard methods known to one skilled in the art.

Any polypeptide demonstrating the desired in vitro activity may then betested in an in vivo animal challenge model. In certain embodiments,immunogenic compositions are used in the immunization of an animal(e.g., a mouse) by methods and routes of immunization known to those ofskill in the art (e.g., intranasal, parenteral, intramuscular, oral,rectal, vaginal, transdermal, intraperitoneal, intravenous,subcutaneous, etc.). Following immunization of the animal with aparticular Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisimmunogenic composition, the animal is challenged with the same or otherstreptococcal species and assayed for resistance to the same or otherStreptococcus spp. infection.

The Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptides and polynucleotides are incorporated intoimmunogenic compositions suitable for administration to a subject, e.g.,a human. Such compositions typically comprise the nucleic acid moleculeor protein, together with a pharmaceutically acceptable carrier. As usedhereinafter the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, excipients and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions.

An immunogenic composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral (e.g., intramuscular, intravenous,intradermal, subcutaneous, intraperitoneal), transmucosal (e.g., oral,rectal, intranasal, vaginal, respiratory) and transdermal (topical).Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and the like. In many cases,isotonic agents are included, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis ORF1358 polypeptide or antibody thereto) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation (Mountain View,Calif.) and Nova Pharmaceuticals, Inc. (Baltimore, Md.). Liposomalsuspensions (including liposomes targeted to infected cells withmonoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Combination immunogenic compositions are provided by combining one ormore of the polypeptides of the invention with one or more knownstreptococcal polysaccharides or polysaccharide-protein conjugates.

The protein component of the carbohydrate-protein conjugates is known asa “carrier protein”. The term “carrier proteins”, includes thoseproteins that are non-toxic, non-reactogenic and obtainable insufficient amount and purity. Carrier proteins are amenable to standardconjugation procedures. For example, CRM₁₉₇ can be used as the carrierprotein. CRM₁₉₇, (Wyeth, Sanford, N.C.) is a non-toxic variant (toxoid)of diphtheria toxin isolated from cultures of Corynebacterium diphtheriastrain C7 (β3197) grown in casamino acids and yeast extract-basedmedium. CRM₁₉₇ is purified through ultra-filtration, ammonium sulfateprecipitation, and ion-exchange chromatography. Other diphtheria toxoidsare also suitable for use as carrier proteins.

Other suitable carrier proteins include inactivated bacterial toxinssuch as tetanus toxoid, pertussis toxoid, cholera toxoid (as describede.g. in PCT Publication No. WO/2004/083251), E. coli LT, E. coli ST, andexotoxin A from Pseudomonas aeruginosa. Bacterial outer membraneproteins such as outer membrane complex c (OMPC), porins, transferrinbinding proteins, pneumolysis, pneumococcal surface protein A (PspA),pneumococcal adhesin protein (PsaA), or Haemophilus influenzae proteinD, can also be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD) can also be used as carrier proteins.

Immunogenic compositions comprising Streptococcus dysgalactiae subsp.equisimilis, Streptococcus intermedius, Streptococcus constellatussubsp. constellatus, Streptococcus anginosus, or Streptococcusconstellatus subsp. pharyngis ORF1358 polynucleotides are delivered tothe recipient by a variety of vectors and expression systems. Suchsystems include, among others, chromosomal, episomal and virus-derivedsystems as mentioned above.

An immunogenic composition of the present invention is typicallyadministered parenterally in unit dosage formulations containingstandard, well-known nontoxic physiologically acceptable carriers,adjuvants, and vehicles as desired.

A pharmaceutically acceptable vehicle is understood to designate acompound or a combination of compounds entering into a pharmaceutical orimmunogenic composition which does not cause side effects and whichmakes it possible, for example, to facilitate the administration of theactive compound, to increase its life and/or its efficacy in the body,to increase its solubility in solution or alternatively to enhance itspreservation. These pharmaceutically acceptable vehicles are well knownand will be adapted by persons skilled in the art according to thenature and the mode of administration of the active compound chosen.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Carriers include neutral saline solutions buffered with phosphate,lactate, Tris, and the like. When administering viral vectors, onepurifies the vector sufficiently to render it essentially free ofundesirable contaminants, such as defective interfering adenovirusparticles or endotoxins and other pyrogens such that it does not causeany untoward reactions in the individual receiving the vector construct.In some embodiments, the means of purifying the vector involves the useof buoyant density gradients, such as cesium chloride gradientcentrifugation.

A carrier can also be a liposome. Means for using liposomes as deliveryvehicles are well known in the art (see, e.g. the review by SchwendenerR A, Adv. Exp. Med. Biol. 620:117-128, 2007)

The immunogenic compositions of this invention also comprise apolynucleotide sequence of this invention operably linked to aregulatory sequence that controls gene expression. The polynucleotidesequence of interest is engineered into an expression vector, such as aplasmid, under the control of regulatory elements that will promoteexpression of the DNA, that is, promoter and/or enhancer elements. Insome embodiments, the human cytomegalovirus immediate-earlypromoter/enhancer is used (U.S. Pat. No. 5,168,062). The promoter may becell-specific and permit substantial transcription of the polynucleotideonly in predetermined cells.

The polynucleotides of the invention are introduced directly into thehost either as “naked” DNA (U.S. Pat. No. 5,580,859) or formulated incompositions with facilitating agents, such as bupivacaine and otherlocal anesthetics (U.S. Pat. No. 5,593,972) and cationic polyamines(U.S. Pat. No. 6,127,170). In this polynucleotide immunizationprocedure, the polypeptides of the invention are expressed on atransient basis in vivo; no genetic material is inserted or integratedinto the chromosomes of the host. This procedure is to be distinguishedfrom gene therapy, where the goal is to insert or integrate the geneticmaterial of interest into the chromosome. An assay is used to confirmthat the polynucleotides administered by immunization do not give riseto a transformed phenotype in the host (e.g., U.S. Pat. No. 6,168,918).

Immunogenic compositions as described herein also comprise, in certainembodiments, one or more adjuvants. An adjuvant is a substance thatenhances the immune response when administered together with animmunogen or antigen. A number of cytokines or lymphokines have beenshown to have immune modulating activity, and thus are useful asadjuvants, including, but not limited to, the interleukins 1-α, 1-β, 2,4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15,16, 17 and 18 (and its mutant forms); the interferons-α, β and γ;granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g.,U.S. Pat. No. 5,078,996 and ATCC Accession Number 39900); macrophagecolony stimulating factor (M-CSF); granulocyte colony stimulating factor(G-CSF); and the tumor necrosis factors α and β. Still other adjuvantsthat are useful with the immunogenic compositions described hereininclude chemokines, including without limitation, MCP-1, MIP-1α, MIP-1β,and RANTES; adhesion molecules, such as a selectin, e.g., L-selectin,P-selectin and E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1and MadCAM-1; a member of the integrin family such as LFA-1, VLA-1,Mac-1 and p150.95; a member of the immunoglobulin superfamily such asPECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3;co-stimulatory molecules such as CD40 and CD40L; growth factorsincluding vascular growth factor, nerve growth factor, fibroblast growthfactor, epidermal growth factor, B7.2, PDGF, BL-1, and vascularendothelial growth factor; receptor molecules including Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspase (ICE).

Suitable adjuvants used to enhance an immune response further include,without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa,Hamilton, Mont.), which is described in U.S. Pat. No. 4,912,094. Alsosuitable for use as adjuvants are synthetic lipid A analogs oraminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa (Hamilton, Mont.), andwhich are described in U.S. Pat. No. 6,113,918. One such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529). This 529 adjuvantis formulated as an aqueous form (AF) or as a stable emulsion (SE).

Still other adjuvants include muramyl peptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE); oil-in-water emulsions, such as MF59 (U.S. Pat. No. 6,299,884)(containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.)), and SAF (containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion); incomplete Freund's adjuvant (IFA);aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate; Amphigen; Avridine; L121/squalene;D-lactide-polylactide/glycoside; pluronic polyols; killed Bordetella;saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.),described in U.S. Pat. No. 5,057,540, ISCOMATRIX (CSL Limited,Parkville, Australia), described in U.S. Pat. No. 5,254,339, andimmunostimulating complexes (ISCOMS); Mycobacterium tuberculosis;bacterial lipopolysaccharides; synthetic polynucleotides such asoligonucleotides containing a CpG motif (e.g., U.S. Pat. No. 6,207,646);IC-31 (Intercell AG, Vienna, Austria), described in European Patent Nos.1,296,713 and 1,326,634; a pertussis toxin (PT) or mutant thereof, acholera toxin or mutant thereof (e.g., U.S. Pat. Nos. 7,285,281,7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin(LT) or mutant thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat.Nos. 6,149,919, 7,115,730 and 7,291,588).

The present invention is directed inter alia to treatment ofstreptococcal infection by administration of therapeutic immunologicalreagents such as humanized monoclonal antibodies recognizing specificepitopes within a Streptococcus dysgalactiae subsp. equisimilis,Streptococcus intermedius, Streptococcus constellatus subsp.constellatus, Streptococcus anginosus, or Streptococcus constellatussubsp. pharyngis ORF1358 polypeptide to a subject under conditions thatgenerate a beneficial therapeutic response in the subject.“Immunological reagents” include e.g. antibodies, humanized antibodies,antibody fragments, peptides comprising antigen binding elements orCDRs, and the like. “Beneficial therapeutic responses” include e.g.induction of phagocytosis or opsonization of beta-hemolyticstreptococci. The invention is also directed to use of the disclosedimmunological reagents in the manufacture of a medicament for thetreatment or prevention of a beta-hemolytic streptococcal infection.

In one aspect, the invention provides methods of preventing or treatingdisease associated with beta-hemolytic streptococcal infection in apatient. Some methods of the invention entail administering to a patientan effective dosage of an antibody that specifically binds to aStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 epitope. Such methods are particularly useful for preventing ortreating beta-hemolytic streptococcal disease in subjects. “Subjects”include any vertebrate animal, such as companion animals, farm animals,mammals, and human patients. Exemplary methods include administering aneffective dosage of an antibody or antigen binding peptide that binds toa Streptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptide. Some embodiments include administering an effectivedosage of an antibody or other peptide comprising an antigen recognitionsite or CDR that specifically binds to an epitope within a Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide, suchas e.g. a polypeptide comprising an amino acid sequence of any one ormore of SEQ ID NOs: 2, 4, 6, 8, 10,12, 13, 26, 28, 30, or 32.

In yet another aspect, the invention features administering antibodiesor other antigen binding peptides that bind to a Streptococcusdysgalactiae subsp. equisimilis, Streptococcus intermedius,Streptococcus constellatus subsp. constellatus, Streptococcus anginosus,or Streptococcus constellatus subsp. pharyngis ORF1358 polypeptide inthe subject and induce a clearing response against a beta-hemolyticstreptococcus. For example, such a clearing response can be effected byFc receptor mediated phagocytosis.

Therapeutic immunological reagents of the invention are typicallysubstantially pure from undesired contaminants. This means that animmunological reagent is typically at least about 50% w/w(weight/weight) purity, as well as being substantially free frominterfering proteins and contaminants. In some embodiments, theimmunological reagents are at least about 80% w/w purity. In otherembodiments, the immunological reagents are at least 90 or about 95% w/wpurity. However, using conventional protein purification techniques,homogeneous peptides of at least 99% w/w purity can be obtained.

The methods can be used on both asymptomatic subjects and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments, peptides comprisingepitope binding regions or CDRs) and can be monoclonal or polyclonal, asdescribed herein.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a subject by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the patient. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thepatient. In exemplary embodiments, the patient is monitored for level ofadministered antibody in the blood of the patient.

Subjects amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. Therefore, the present immunogenic compositions andtherapeutic antibodies can be administered prophylactically to thegeneral population. In asymptomatic subjects, treatment can begin at anyage. Treatment can be monitored by assaying antibody levels over time.If the immune response or antibody level falls, a booster dosage isindicated.

In prophylactic applications, immunogenic compositions or medicamentsare administered to a subject susceptible to, or otherwise at risk of,beta-hemolytic streptococcal infection in an amount sufficient toeliminate or reduce the risk, lessen the severity, or delay the outsetof the disease, including biochemical, histological and/or behavioralsymptoms of disease associated with the infection, its complications andintermediate pathological phenotypes presenting during development ofthe disease. In therapeutic applications, compositions or medicamentsare administered to a patient suspected of, or already suffering fromsuch a disease in an amount sufficient to cure, or at least partiallyarrest, the symptoms of the disease (biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of the disease.

An amount adequate to accomplish therapeutic or prophylactic treatmentis defined as a therapeutically- or prophylactically-effective dose. Inboth prophylactic and therapeutic regimes, immunological reagents areusually administered in several dosages until a sufficient immuneresponse has been achieved. The term “immune response” or “immunologicalresponse” includes the development of a humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against an antigen in a recipientsubject. Such a response can be an active response, i.e., induced byadministration of immunogen (supra), or a passive response, i.e.,induced by administration of immunoglobulin or antibody or primedT-cells. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of beta-hemolytic streptococcal infection vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, whether the patient is human oranother animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the subject is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may need to be titrated to optimize safety andefficacy.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg,0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe about 1 mg/kg body weight or about 10 mg/kg body weight or within therange of 1 to 10 mg/kg. Doses intermediate in the above ranges are alsointended to be within the scope of the invention. Subjects can beadministered such doses daily, on alternative days, weekly, monthly,every two months, every three months, or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. Additional exemplary treatment regimes entailadministration once per every two weeks or once a month or once every 3to 6 months. Exemplary dosage schedules include 1 to 10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toStreptococcus dysgalactiae subsp. equisimilis, Streptococcusintermedius, Streptococcus constellatus subsp. constellatus,Streptococcus anginosus, or Streptococcus constellatus subsp. pharyngisORF1358 polypeptide in the patient. In some methods, dosage is adjustedto achieve a plasma antibody concentration of 1 to 1000 pg/ml and insome methods 25 to 300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanizedantibodies show the longest half-life, followed by chimeric antibodiesand nonhuman antibodies

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 pg to 10 mg, or 30 to 300 pg DNA per patient.Doses for infectious viral vectors vary from 10 to 100, or more, virionsper dose.

Therapeutic immunological reagents can be administered by parenteral,topical, intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical routes of administrationof an immunogenic agent are intravenous infusion or subcutaneousadministration, although other routes can be equally effective. The nextmost common route is intramuscular injection. This type of injection ismost typically performed in the arm or leg muscles. In some methods,immunological reagents are injected directly into a particular tissuewhere deposits have accumulated, for example intracranial injection.Intramuscular injection or intravenous infusion are preferred foradministration of antibody. In some methods, antibodies are administeredas a sustained release composition or device, such as a microinfusordevice (e.g. Medipad™ device; see Meehan et al., Journal of ControlledRelease, 46:107-119, 1997.)

As alluded to above, immune responses against beta-hemolyticstreptococcal infection can be formed in vivo (or ex vivo) byadministration of nucleic acids encoding antibodies and their componentchains used for passive immunization. Such nucleic acids can be DNA orRNA. A nucleic acid segment encoding an immunological reagent istypically linked to regulatory elements, such as a promoter andenhancer, that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of an immune response, promoter and enhancerelements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to directexpression. The linked regulatory elements and coding sequences areoften cloned into a vector. For administration of double-chainantibodies, the two chains can be cloned in the same or separatevectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3:102109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67:5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179:1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508, 1996),Venezuelan equine encephalitis virus (see Johnston et al., U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (seeRose, U.S. Pat. No. 6,168,943) and papillomaviruses (Ohe et al., HumanGene Therapy 6:325, 1995; Woo et al., PCT publication No. WO 94/12629and Xiao and Brandsma, Nucleic Acids. Res. 24: 2630-2622, 1996).

DNA encoding an antibody or antibody fragment comprising a CDR, or avector containing the same, can be packaged into liposomes. Suitablelipids and related analogs are described by Eppstein et al., U.S. Pat.No. 5,208,036, Felgner et al., U.S. Pat. No. 5,264,618, Rose, U.S. Pat.No. 5,279,833, and Epand et al., U.S. Pat. No. 5,283,185. Vectors andDNA encoding an immunogen can also be adsorbed to or associated withparticulate carriers, examples of which include polymethyl methacrylatepolymers and polylactides and poly (lactide-co-glycolides), see, e.g.,McGee et al., J. Microencapsul. 14(2):197-210, 1997.

Polynucleotide vectors or naked polynucleotides (e.g., DNA) can bedelivered in vivo by administration to an individual patient, typicallyby systemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide which is not administered together with a transfectionfacilitating agent. Naked polynucleotides are sometimes cloned in aplasmid vector. Plasmid vectors can further include transfectionfacilitating agents such as bupivacaine (Weiner et al., U.S. Pat. No.5,593,972). DNA can also be administered using a gene gun. See Xiao andBrandsma, supra. The DNA encoding an antibody (or fragment comprising aCDR) is precipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The ACCEL™ Gene Delivery Device, i.e., a DNA gun, manufacturedby Agricetus, Inc. Middleton Wis. is suitable for use in the practice ofthis invention. Alternatively, naked DNA can pass through skin into theblood stream simply by spotting the DNA onto skin with chemical ormechanical irritation (see Howell et al., PCT Publication No. WO95/05853).

In another embodiment, vectors encoding immunological reagents can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

Immunological reagents of the invention can optionally be administeredin combination with other agents that are at least partly effective intreatment of beta-hemolyic streptococcal disease. Immunological reagentsof the invention can also be administered in combination with otheragents that enhance access of the therapeutic immunological reagent to atarget cell or tissue, for example, liposomes and the like.Coadministering such agents can decrease the dosage of a therapeuticimmunological reagent (e.g., therapeutic antibody or antibody chain)needed to achieve a desired effect.

Immunological reagents of the invention are often administered aspharmaceutical compositions comprising an active therapeutic agent,i.e., and a variety of other pharmaceutically acceptable components. SeeRemington's Pharmaceutical Science (15th ed., Mack Publishing Company,Easton, Pa., 1980). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, immunological reagents of the inventioncan be administered as injectable dosages of a solution or suspension ofthe substance in a physiologically acceptable diluent with apharmaceutical carrier that can be a sterile liquid such as water oils,saline, glycerol, or ethanol. Additionally, auxiliary substances, suchas wetting or emulsifying agents, surfactants, pH buffering substancesand the like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). Theimmunological reagents of this invention can be administered in the formof a depot injection or implant preparation, which can be formulated insuch a manner as to permit a sustained or pulsatile release of theactive ingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1 % to 2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10% to 95% of active ingredient,preferably 25% to 70%.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes (Paul et al., Eur. J. Immunol. 25:3521, 1995;Cevc et al., Biochem. Biophys. Acta 1368:201-215, 1998).

The invention also provides methods of monitoring treatment in a patientsuffering from or susceptible to beta-hemolytic streptococcal infection,i.e., for monitoring a course of treatment being administered to apatient. The methods can be used to monitor both therapeutic treatmenton symptomatic patients and prophylactic treatment on asymptomaticpatients. In particular, the methods are useful for monitoring passiveimmunization (e.g., measuring level of administered antibody).

Some methods entail determining a baseline value, for example, of anantibody level or profile in a patient, before administering a dosage ofimmunological reagent, and comparing this with a value for the profileor level after treatment. A significant increase (i.e., greater than thetypical margin of experimental error in repeat measurements of the samesample, expressed as one standard deviation from the mean of suchmeasurements) in value of the level or profile signals a positivetreatment outcome (i.e., that administration of the immunologicalreagent has achieved a desired response). If the value for immuneresponse does not change significantly, or decreases, a negativetreatment outcome is indicated. If the treatment is passiveimmunotherapy, the antibody level is expected to decrease over time witha characteristic half-life.

The tissue sample for analysis is typically blood, plasma, serum, mucousfluid or cerebrospinal fluid from the patient. The sample is analyzed,for example, for levels or titers of antibodies to streptococcal PPI.ELISA methods of detecting antibodies specific to streptococcal PPI aredescribed in the Examples section. In some methods, the level or titerof an administered antibody is determined using a clearing assay, forexample, in an in vitro phagocytosis assay (see, e.g., Jansen et al.,Clin. Diagn. Lab. Immunol., 8(2): 245-250, 2001.)

The antibody profile following passive immunization typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered.

In some methods, a baseline measurement of antibody to streptococcal PPIin the patient is made before administration, a second measurement ismade soon thereafter to determine the peak antibody level, and one ormore further measurements are made at intervals to monitor decay ofantibody levels. When the level of antibody has declined to baseline ora predetermined percentage of the peak less baseline (e.g., 50%, 25% or10%), administration of a further dosage of antibody is administered. Insome methods, peak or subsequent measured levels less background arecompared with reference levels previously determined to constitute abeneficial prophylactic or therapeutic treatment regime in otherpatients. If the measured antibody level is significantly less than areference level (e.g., less than the mean minus one standard deviationof the reference value in population of patients benefiting fromtreatment) administration of an additional dosage of antibody isindicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom routinely relied on by researchers orphysicians to diagnose or monitor streptococcal infections or associateddiseases. For example, one can monitor symptoms of cellulitis,erysipelas, impetigo, necrotizing fasciitis, sore throat, red throat,chills, fever, headache, nausea, vomiting, rapid heartbeat, malaise,swollen tonsils. enlarged lymph nodes and/or rash.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

EXAMPLES Example 1 Identification of ORF1358 in Streptococcal Strains

The DNA and protein sequences of Streptococcal candidate antigens havebeen identified in many of streptococcal genomes. However, limitedsequence information exists on the Group C and G Streptococcal genomes.Currently, two animal-origin streptococcal genomes (Group C) are beingsequenced by the Sanger Centre, Streptococcus equi and Streptococcuszooepidemicus. Data mining of the partially finished genomes usingdegenerate primers yielded DNA sequences of ORF1358.

Degenerate oligonucleotide probes were designed to identify ORF1358 inStreptococcus dysgalactiae subsp. equisimilis strain ATCC12394 andstrain ATCC35666, Streptococcus intermedius strain ATCC27335,Streptococcus constellatus subsp. constellatus strain ATCC27823,Streptococcus anginosus strain ATCC33397, and Streptococcus constellatussubsp. pharyngis strain NTCT13122.

All known sequences containing ORF1358 were aligned using AlignX (VectorNTI) and regions of homology were used for degenerate primerconstruction. Primers were designed to have minimal degeneracy whilemaintaining a high melting temperature and low self dimerizationpotential. The nucleotide sequences of the primers are set forth in SEQID NOs:14 through 21.

Primers were analyzed using the website for Integrated DNA Technologies(Coraville, Iowa). Initial amplification studies were performed usinggenomic DNA preparations made to a Streptococcus C isolate ATCC12394(Streptococcus dysgalactiae supsp. equisimilis). Partial gene sequenceswere obtained to the 5′ and 3′ of ORF1358. Forward and reverse primerswere then designed based on these sequences and were subsequently usedto amplify approximately 700-900 bp of sequence from ORF1358 fromdifferent G and C strains. The primers based on ATCC12394 are set forthin SEQ ID NO:20 and 21.

Using methods familiar to those skilled in the art, genomic DNA wasprepared from each of the strains mentioned above, and polynucleotidefragments corresponding to ORF1358 were amplified using the primers setforth in SEQ ID NO:14-21. The nucleotide sequences obtained for ORF1358are set forth in SEQ ID NOs:1, 3, 5, 7, 9, 11, 25, 27, 29, and 31.

Translation of these polynucleotides resulted in the amino acidsequences of ORF1358 in these Streptococcus strains. The polynucleotidesequences are set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 26, 28, 30,and 32.

The amino acid sequences in SEQ ID NOs:2, 4, 6, 8, 10, and 12 werealigned using Clustal W, and a consensus sequence generated and setforth in SEQ ID NO:13.

Table 1A depicts the pair distances obtained using ClustalW(slow/accurate, Gonnet) or percent identity obtained after aligning theORF1358 amino acid sequences set forth in SEQ ID NOs:2,4,6,8,10, and 12with the ORF1358 amino acid sequences set forth in SEQ ID NOs:22, 23,and 24:

TABLE 1A Streptococcal Percent Identities SEQ SEQ SEQ ID NO: 22 ID NO:23 ID NO: 24 SEQ ID NO: 2 96.6 72.9 87.1 SEQ ID NO: 4 96.5 72.9 87.7 SEQID NO: 6 96.9 73.6 87.0 SEQ ID NO: 8 97.2 73.5 87.3 SEQ ID NO: 10 97.073.5 87.1 SEQ ID NO: 12 97.3 74.0 87.2

Table 1B depicts the pair distances or percent identity obtained usingBLAST after aligning the ORF1358 amino acid sequences set forth in SEQID NOs:2,4,6,8,10, 12, 26, 28, 30, and 32 with the ORF1358 amino acidsequences set forth in SEQ ID NOs:22 and 23:

TABLE 1B Streptococcal Percent Identities SEQ ID NO: 22 SEQ ID NO: 23SEQ ID NO: 2 96 71 SEQ ID NO: 4 96 71 SEQ ID NO: 6 97 70 SEQ ID NO: 8 9770 SEQ ID NO: 10 96 70 SEQ ID NO: 12 97 71 SEQ ID NO: 26 96 69 SEQ IDNO: 28 96 70 SEQ ID NO: 30 61 62 SEQ ID NO: 32 61 61

Example 2 Antibodies to Group C/G Staphylococcal ORF 1358 Epitopes

The binding of antibodies to bacteria, a process known as opsonization,can lead to uptake and killing of the bacteria by phagocytic cells. Suchantibodies, whether derived from bulk human or animal sources, or humanor murine or chimeric monoclonal sources, and used alone or incombination, could be used in either prophylactic or therapeuticsettings where BHS might be present in the bloodstream, such as neonatalsepsis or sepsis following surgery or leaking of an abscess.

Antibodies were raised in mice against recombinant group C/Gstaphylococcal zinc binding polypeptides encoded by ORF1358. In thecourse of screening those anti-beta-hemolytic-streptococcal antisera andmonoclonal antibodies against various beta-hemolytic streptococcal (BHS)strains, it was noted that some antisera and antibodies arecross-reactive against many BHS strains, including members ofStreptococcus pyogenes (Group A streptococci), Streptococcus agalactiae(Group B streptococci) and Group C and Group G streptococci (whichinclude the streptococcal species Streptococcus anginosus, Streptococcusconstellatus, Streptococcus intermedius, Streptococcus dysgalactiae sub.Equisimilis and Streptococcus dysgalactiae sub. Dysgalactiae) (See Table1). Screening of the antibodies was performed using fluorescenceactivated cell sorting (FACS). Briefly, heat killed streptococci wereincubated with a mouse anti-Group C and Group G ORF1358 streptococcalantibody on ice for 45 minutes, followed by two 10% FBS/PBS washes. Thestreptococci were then incubated with a goat anti-mouse-Alexa-488antibody (Molecular Probes, Eugene, Oreg.) for 30 minutes on ice,followed by two 10% FBS/PBS washes. Cells were then resuspended in 10%FBS/PBS and run on a FACS machine (e.g. see DeMaster et al., Infect.Immun., 70(1): 350-359, 2002.) This cross-reactivity also means thatGroup C or Group G ORF1358 or the polypeptide encoded thereby may beused in an immunogenic composition to induce an immune responseeffective to protect against infection by Group A or Group BStreptococcus, as well as by Group C or Group G Streptococcus.

Table 2 depicts the cross reactivity of anti-sera and antibodies to thegroup c/g streptococcal polypeptide encoded by ORF1358. According toTable 2, the symbol “+” means that the signal obtained from the specificantibody to the antigen is at least three-fold higher than background;the symbol “±” means that the signal obtained from the specific antibodyto the antigen is between two-fold and three-fold higher thanbackground; and the symbol “−” means that the signal obtained from thespecific antibody to the antigen is at or below background.

TABLE 2 ANTIBODY CROSS-REACTIVITY Reactivity to Strain Species anti-ORF1358 GAR 1165 Streptococcus pyogenes + GAR 1199 Streptococcus pyogenes +GAR 1251 Streptococcus pyogenes + GAR 1278 Streptococcus pyogenes + GAR1362 Streptococcus pyogenes + GAR 1439 Streptococcus pyogenes + GAR 1530Streptococcus pyogenes + GAR 1566 Streptococcus pyogenes + GAR 1672Streptococcus pyogenes + GAR 1839 Streptococcus pyogenes + GAR 1923Streptococcus pyogenes + GAR 2107 Streptococcus pyogenes + GAR 2330Streptococcus pyogenes + GAR 2646 Streptococcus pyogenes + GAR 2650Streptococcus pyogenes + GAR 2869 Streptococcus pyogenes + GAR 3104Streptococcus pyogenes + GAR 3549 Streptococcus pyogenes + GAR 3784Streptococcus pyogenes + GAR 4029 Streptococcus pyogenes + GAR 4030Streptococcus pyogenes + GAR 4230 Streptococcus pyogenes + GAR 4773Streptococcus pyogenes + GAR 4983 Streptococcus pyogenes + GAR 4987Streptococcus pyogenes + GAR 5861 Streptococcus pyogenes + GAR 5991Streptococcus pyogenes + GAR 6084 Streptococcus pyogenes + GAR 7055Streptococcus pyogenes + GS20 Streptococcus pyogenes + GS21Streptococcus pyogenes + GS22 Streptococcus pyogenes + GS23Streptococcus pyogenes + GS24 Streptococcus pyogenes + GS25Streptococcus pyogenes + GS26 Streptococcus pyogenes + GS27Streptococcus pyogenes + GS28 Streptococcus pyogenes + GS29Streptococcus pyogenes + GS30 Streptococcus pyogenes + GS31Streptococcus pyogenes +/− GS32 Streptococcus pyogenes + GS33Streptococcus pyogenes + GS34 Streptococcus pyogenes + GS35Streptococcus pyogenes + GS36 Streptococcus pyogenes +/− GS37Streptococcus pyogenes + GS38 Streptococcus pyogenes + GS39Streptococcus pyogenes + GS40 Streptococcus pyogenes + GS41Streptococcus pyogenes + GS42 Streptococcus pyogenes +/− GS43Streptococcus pyogenes + GS44 Streptococcus pyogenes + GS45Streptococcus pyogenes + GS46 Streptococcus pyogenes + GS47Streptococcus pyogenes +/− GS 48 Streptococcus pyogenes +/− GS 49Streptococcus pyogenes + GS 50 Streptococcus pyogenes + GS 51Streptococcus pyogenes + GS 52 Streptococcus pyogenes + GS 53Streptococcus pyogenes + GS 54 Streptococcus pyogenes +/− GS 55Streptococcus pyogenes + GS 56 Streptococcus pyogenes + GS 57Streptococcus pyogenes + GS 58 Streptococcus pyogenes + GS 59Streptococcus pyogenes + GS 60 Streptococcus pyogenes + GS 61Streptococcus pyogenes + GS 62 Streptococcus pyogenes + GS 63Streptococcus pyogenes + GS 64 Streptococcus pyogenes + GS 65Streptococcus pyogenes + GS 66 Streptococcus pyogenes + GAR 1Streptococcus agalactiae + GAR 1012 Streptococcus agalactiae − GAR 1023Streptococcus agalactiae − GAR 1049 Streptococcus agalactiae − GAR 10895Streptococcus agalactiae − GAR 1192 Streptococcus agalactiae +/− GAR 127Streptococcus agalactiae − GAR 12790 Streptococcus agalactiae − GAR 1305Streptococcus agalactiae − GAR 131 Streptococcus agalactiae − GAR 1355Streptococcus agalactiae − GAR 1446 Streptococcus agalactiae − GAR 1494Streptococcus agalactiae − GAR 154 Streptococcus agalactiae + GAR 176Streptococcus agalactiae − GAR 18 Streptococcus agalactiae + GAR 1844Streptococcus agalactiae − GAR 1931 Streptococcus agalactiae − GAR 2369Streptococcus agalactiae +/− GAR 252 Streptococcus agalactiae − GAR 2533Streptococcus agalactiae − GAR 2682 Streptococcus agalactiae − GAR 2717Streptococcus agalactiae − GAR 2723 Streptococcus agalactiae − GAR 2724Streptococcus agalactiae − GAR 2842 Streptococcus agalactiae − GAR 287Streptococcus agalactiae − GAR 3003 Streptococcus agalactiae − GAR 3751Streptococcus agalactiae − GAR 381 Streptococcus agalactiae − GAR 3830Streptococcus agalactiae − GAR 4131 Streptococcus agalactiae − GAR 4293Streptococcus agalactiae +/− GAR 4398 Streptococcus agalactiae − GAR 462Streptococcus agalactiae − GAR 4837 Streptococcus agalactiae − GAR 54Streptococcus agalactiae − GAR 562 Streptococcus agalactiae + GAR 6016Streptococcus agalactiae + GAR 614 Streptococcus agalactiae +/− GAR 63Streptococcus agalactiae + GAR 6332 Streptococcus agalactiae + GAR 6387Streptococcus agalactiae +/− GAR 6505 Streptococcus agalactiae +/− GAR67 Streptococcus agalactiae − GAR 864 Streptococcus agalactiae +/− GAR967 Streptococcus agalactiae − GS19 GGS +/− GS27 GGS +/− ATCC 33397Streptococcus anginosus +/− ATCC 33397 Streptococcus anginosus − GAR10823 Streptococcus anginosus +/− GAR 1272 Streptococcus anginosus − GAR1370 Streptococcus anginosus − GAR 1425 Streptococcus anginosus +/− GAR1592 Streptococcus anginosus − GAR 1595 Streptococcus anginosus − GAR2044 Streptococcus anginosus − GAR 2523 Streptococcus anginosus − GAR2565 Streptococcus anginosus − GAR 2697 Streptococcus anginosus +/− GAR2822 Streptococcus anginosus − GAR 3091 Streptococcus anginosus − GAR3560 Streptococcus anginosus + GAR 3576 Streptococcus anginosus +/− GAR3858 Streptococcus anginosus +/− GAR 3938 Streptococcus anginosus − GAR4133 Streptococcus anginosus +/− GAR 4158 Streptococcus anginosus + GAR4234 Streptococcus anginosus + GAR 4426 Streptococcus anginosus + GAR4680 Streptococcus anginosus + GAR 4834 Streptococcus anginosus +/− GAR4896 Streptococcus anginosus + GAR 5093 Streptococcus anginosus + GAR5094 Streptococcus anginosus + GAR 5675 Streptococcus anginosus − GAR5776 Streptococcus anginosus + GAR 5831 Streptococcus anginosus +/− GAR6187 Streptococcus anginosus +/− GAR 6590 Streptococcus anginosus +/−GAR 7000 Streptococcus anginosus +/− GAR 7023 Streptococcus anginosus −GAR 7190 Streptococcus anginosus − GAR 7214 Streptococcus anginosus +/−GAR 7468 Streptococcus anginosus − GAR 7818 Streptococcus anginosus +GAR 8620 Streptococcus anginosus + GAR 8693 Streptococcus anginosus −GAR 8722 Streptococcus anginosus +/− GAR 8736 Streptococcus anginosus −GAR 8954 Streptococcus anginosus +/− ATCC 27823 Streptococcusconstellatus − GAR 1235 Streptococcus constellatus − GAR 1384Streptococcus constellatus +/− GAR 1811 Streptococcus constellatus + GAR2421 Streptococcus constellatus +/− GAR 3145 Streptococcus constellatus− GAR 3355 Streptococcus constellatus − GAR 4048 Streptococcusconstellatus +/− GAR 4083 Streptococcus constellatus + GAR 4861Streptococcus constellatus + GAR 4870 Streptococcus constellatus + GAR5757 Streptococcus constellatus +/− GAR 6129 Streptococcus constellatus+/− GAR 6147 Streptococcus constellatus − GAR 6258 Streptococcusconstellatus + GAR 7224 Streptococcus constellatus + GAR 7369Streptococcus constellatus + ATCC 12394 Streptococcus dysgalactiae +/−ATCC 12394 Streptococcus dysgalactiae + ATCC 40378 Streptococcusdysgalactiae − ATCC 40378 Streptococcus dysgalactiae − GAR 3868Streptococcus dysgalactiae +/− GAR 4272 Streptococcus dysgalactiae +ATCC 35666 Streptococcus dysgalactiae + sub. Equisimilis BAA-338Streptococcus dysgalactiae +/− sub. Equisimilis GAR 3015 Streptococcusequisimilis + ATCC 27335 Streptococcus intermedius + ATCC 27335Streptococcus intermedius + GAR 2407 Streptococcus intermedius − GS28unk + GS67 GGS/GCS + GS68 GGS/GCS +/− GS69 GGS/GCS − GS70 GGS/GCS +/−GS71 GGS/GCS + GS72 GGS/GCS + GS73 GGS/GCS − GS74 GGS/GCS − GS75 GGS/GCS+/− GS77 GGS/GCS + GS78 GGS/GCS + GS79 GGS/GCS +/− GS80 GGS/GCS − GS81GGS/GCS +/− GS82 GGS/GCS +/− GS83 GGS/GCS + GS84 GGS/GCS − GS85 GGS/GCS− GS86 GGS/GCS +/− GS88 GGS/GCS + GS89 GGS/GCS +/− GS90 GGS/GCS +/− GS91GGS/GCS +/− GS92 GGS/GCS + GS93 GGS/GCS + GS94 GGS/GCS +

1. An isolated polypeptide that comprises an amino acid sequence that isat least 95% identical to any one or more of SEQ ID NO: 2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33. 2.The isolated polypeptide of claim 1, wherein the polypeptide comprisesan amino acid sequence that is at least 97% identical to any one or moreof SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,SEQ ID NO:32, and SEQ ID NO:33.
 3. An isolated polynucleotide whichencodes any one of an isolated polypeptide that comprises: a) an aminoacid sequence that is at least 95% identical to any one or more of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, and SEQ ID NO:33; or b) an amino acid sequence of any one of SEQID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, and SEQ ID NO:33.
 4. The isolated polynucleotide of claim 3,wherein the polynucleotide comprises a nucleotide sequence that is atleast 95% identical to any one or more of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, and SEQ ID NO:31.
 5. The isolated polynucleotide ofclaim 3, wherein the polynucleotide comprises a nucleotide sequence thatis at least 97% identical to any one or more of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.
 6. An isolatedpolynucleotide of claim 3, wherein the polynucleotide is operably linkedto a regulatory element.
 7. A polynucleotide vector comprising anisolated polynucleotide that encodes any one of an isolated polypeptidethat comprises: a) an amino acid sequence that is at least 95% identicalto any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and SEQ ID NO:32; or b) an amino acid sequence ofany one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, and SEQ ID NO:32; operably linked to a regulatory element.
 8. Thepolynucleotide vector of claim 7, wherein the polynucleotide vector isany one or more of a plasmid, a viral vector, and an expression vector.9. A cell comprising an isolated polynucleotide which encodes any one ofan isolated polypeptide that comprises: a) an amino acid sequence thatis at least 95% identical to any one or more of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; or b) anamino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32. wherein the cell isex vivo.
 10. The cell of claim 9, wherein the cell is selected from thegroup consisting of a bacterium, a mammalian cell, an insect cell, and ayeast cell.
 11. An immunogenic composition comprising an isolatedpolypeptide that comprises: a) an amino acid sequence that is at least95% identical to any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; or b) an amino acidsequence of any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and SEQ ID NO:32.
 12. An immunogenic compositioncomprising an isolated polynucleotide that encodes a polypeptide thatcomprises: a) an amino acid sequence that is at least 95% identical toany one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, and SEQ ID NO:32; or b) an amino acid sequence of any oneof SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,and SEQ ID NO:32.
 13. A method for inducing an immune response to a betahemolytic Streptococcus bacterium or to a beta hemolytic streptococcalinfection in a patient comprising administering to the patient animmunogenic composition comprising administering to the patient animmunogenic composition comprising: a) a polypeptide that comprises: i)an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; or ii) an aminoacid sequence that is at least 95% identical to any one or more of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:32; or b) a polynucleotide that encodes a polypeptide of (a). 14.The method of claim 14 wherein the beta hemolytic Streptococcusbacterium or the beta hemolytic streptococcal infection is from Group A,Group B, Group C or Group G.
 15. A kit comprising an isolatedpolypeptide that comprises an amino acid sequence that is at least 95%identical to any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, and SEQ ID NO:32.
 16. A kit comprising anisolated polynucleotide that encodes a polypeptide that comprises anamino acid sequence that is at least 97% identical to any one or more ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, andSEQ ID NO:32.
 17. A method of producing an isolated polypeptide, whichcomprises transforming, transfecting or infecting a cell with a plasmidcontaining an isolated polynucleotide which encodes an isolatedpolypeptide comprising an amino acid sequence that is at least 95%identical to any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, and SEQ ID NO:32, and culturing the cell underconditions which permit the expression of said polypeptide by the cell,and purifying said polypeptide from the cell.